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Signaling Dynamics During Vertebrate Segmentation Alexis Hubaud Signaling dynamics during Vertebrate segmentation Alexis Hubaud To cite this version: Alexis Hubaud. Signaling dynamics during Vertebrate segmentation. Embryology and Organogenesis. Université de Strasbourg, 2016. English. NNT : 2016STRAJ101. tel-01548716 HAL Id: tel-01548716 https://tel.archives-ouvertes.fr/tel-01548716 Submitted on 28 Jun 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ IGBMC - CNRS UMR 7104 - Inserm U 964 THÈSE présentée par : Alexis HUBAUD soutenue le : 27 juin 2016 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Biologie du Développement Dynamique de la signalisation cellulaire au cours de la segmentation des Vertébrés THÈSE dirigée par : Mr. POURQUIÉ Olivier Professeur, Université de Strasbourg RAPPORTEURS : Mr. AULEHLA Alexander Professeur, EMBL Heidelberg Mr. PAUL François Professeur, McGill University AUTRES MEMBRES DU JURY : Mme. GUEVORKIAN Karine Chargée de Recherche, Université de Strasbourg Mr. GREGOR Thomas Professeur, Princeton University ACKNOWLEDGMENTS I would like to acknowledge all the people who have contributed to this work and who have accompanied me from my early undergraduate days in Paris to the end of my PhD in Boston. First I would like to thank my teachers, supervisors, and former lab mates, who have helped me and trained me: Myriam Gazeau for her wonderful biology classes, her enthusiasm and efforts making her lectures fascinating, Viviane Campomar, Claire Santoni, Rémi Dejean for their great teaching. I would like to thank my first internship supervisor, Vincent Neyraud for his time and kindness; Julie Mathieu, Hannele Ruohola-Baker, and members of her lab for a very good time in Seattle. I am very grateful to Antonio Giraldez to have welcomed me in his lab and taught me so many things about research. I would like also to thank all the members of his lab, especially Daniel Cifuentes and Carlos Strahlut for their help and guidance. I would like to thank Olivier Pourquié, my PhD supervisor, for his trust, for the opportunity and independence he gave me to work on interesting projects and for the resources I had during my PhD to pursue them. I would like to thank all the members of his lab, past and present. I am especially grateful to Bertrand Bénazéraf, who supervised me during my master and has kindly supported me during my PhD. I am also very grateful to Stéphane Vincent for everything he did during my PhD, I am indebted for all the help, tips and advice he gave me. ࠶ࡾࡀ࡜࠺ࡈࡊ࠸ࡲࡋࡓ to Masayuki Oginuma for all the discussions about science and other crazy things, I would like to thank Jérome Chal for making easier the transition to Boston and for his characteristic enthusiasm, Goncalo Cadete Vilhais Neto for his guidance as a benchmate and the scientific discussions, and all the people of the lab for their help, discussions and support, notably Nicolas Denans, Agata Bera, Marjorie Fournier, Ayako Myanari, Karine Guevorkian, Bénédicte Gobert, Jean-Marie Garnier, Jean-Daniel Fauny, Tania Knauer-Meyer, Olivier Tassy, Mike Rebagliati, Mathias Wahl, Paul Bardot, Arthur Michaut, Charlotte Fugier, Marie Hestin, Getzabel Guevara, Charlène Guillot, Fengzhu Xiong, Suvi Aivio, Marie Knockaert, Adrian Ranga, and Myriam Rebetez. I would like to thank Paul François, Alexander Aulehla, Karine Guevorkian and Thomas Gregor for accepting to be part of my thesis committee, and for their time and effort in evaluating my dissertation. I would also like to thank Andy Oates and Gilles Charvin for having participated to my mid-thesis committee. I would like to thank Camille Paoletti and members of the Charvin lab for their nice help with microfluidics. I would like to thank L.Mahadevan for his enthusiasm and for his valuable input on this work. I would like to thank Charisios Tsiairis, Tom Hiscock, Bryan Hassel, Ido Regev for their help and discussions. I would like to thank the engineers and technicians at the IGBMC and at BWH/HMS, notably Marc Koch and Pascal Kessler at the IGBMC imaging facility, Claudine Ebel at the IGBMC FACS facility, Raphaël Bour and Chadia Toubari at the IGBMC/ICS mice facility. I would like to thank the organizations which granted me fellowships during my studies: the French Ministry of Research and Higher Education, the Fondation pour la Recherche Medicale, the Boehringer-Ingelheim Fonds and the EMBL Corporate Partnership Programme. Last, I would like to thank my family, especially Annick, Benoit, Aude, Raymond, Françoise, and my friends, especially Jean-Marie and Thibaut, for all their support during my PhD. TABLE OF CONTENTS List of Figures ........................................................................................................................1 Introduction ...........................................................................................................................4 1. Overview of Vertebrate Development ......................................................................................... ..6 2. Development of the presomitic mesoderm ......................................................................................9 3. Segmentation and Somitogenesis ..................................................................................................22 4. The segmentation clock .................................................................................................................30 5. Signaling gradients and determination of the PSM .......................................................................47 6. Somitogenesis models ...................................................................................................................63 7. Integrated view of PSM development ...........................................................................................81 Chapter I – Transcriptional regulation of Mesp factors .................................................93 1. Regulation of Mesp2 expression ...................................................................................................94 2. Tools to study the formation of the Fgf8 gradient .......................................................................107 Chapter II – Emergence of oscillations and their molecular control ...........................114 1. Explant system to study the segmentation clock .........................................................................115 2. Culture conditions .......................................................................................................................120 3. Distribution of Fgf signaling .......................................................................................................124 4. Dynamics at the cellular level .....................................................................................................127 5. Existence of a refractory period ..................................................................................................131 6. Role of the Notch pathway ..........................................................................................................131 7. Role of mechanical factors and the Yap pathway .......................................................................137 8. Role of Fgf signaling in the PSM determination .........................................................................145 9. Role of Wnt signaling in the PSM determination .......................................................................154 Discussion .......................................................................................................................................158 Chapter III – Role of the translation rate on the segmentation clock ..........................175 1. Effect of the translation inhibitor cycloheximide ........................................................................176 2. Regulation of the translation ......................................................................................................176 3. Visualization of protein synthesis ...............................................................................................178 Discussion .......................................................................................................................................180 Chapter IV – Reconstituting the segmentation clock in vitro .......................................184 1. Derivation and characterization of a LuVeLu mESC line ...........................................................185 2. Creation of a LuVeLu/Msgn1-mCherry cell line .........................................................................187 3. Conditions for the apparition of oscillations ...............................................................................192 4. Generation of alternative cyclic and cell fate reporters ...............................................................194 5. Control of signaling gradients by microfluidics ..........................................................................198
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